This invention relates to the fields of genomics and pharmacogenetics. More specifically, the present invention relates to polymorphisms and haplotypes of the xcex22-adrenergic receptor gene and their use as predictors of disease susceptibility and response to xcex2-agonists.
The xcex22-adrenergic receptor (xcex22AR) is a G protein coupled receptor that mediates the actions of catecholamines in multiple tissues and thus plays important roles in regulating cardiac, vascular, pulmonary, and metabolic functions. An abnormal level of expression of xcex22AR is believed to be a risk factor for or to modify the severity of a number of diseases and conditions, including congestive heart failure, arrhythmia, ischemic heart disease, hypertension, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes, myasthenia gravis, and premature labor.
The xcex22AR is encoded by an intronless gene on chromosome 5q31-32 Kobilka, B. K. et al., Proc. Natl. Acad. Sci., USA 84:46-50, 1987). Several single nucleotide polymorphisms (SNPs) in the coding block of the xcex22AR gene that lead to significant genetic variability in the structure of the xcex22AR protein in the human population have been reported (Reihsaus, E. et al., Am J Resp Cell Mol Biol 8:334-339, 1993; Liggett, S. B., News in Physiologic Sciences 10:265-273, 1995; and GenBank accession numbers AF022953.1 GI:2570526; AF022954.1 GI:2570528; and AF022956.1 GI:2570532). These SNPs are located at nucleotides 46 (A or G), 79 (C or G), and 491 (C or T) of the xcex22AR coding sequence, and result in variation that occurs in the amino-terminus of the receptor at amino acids 16 (Arg or Gly) and 27 (Gln or Glu) and in the fourth transmembrane spanning domain at amino acid 164 (Thr or Ile), respectively. These amino acid variants have clear phenotypic differences as demonstrated by recombinant cell studies (Green, S. A. et al., Biochem 33:9414-9419, 1994; Green, S. A., et al., J Biol Chem 268:23116-23121, 1993), primary cultures of cells endogenously expressing these variants (Green, S. A., et al., Am J Resp Cell Mol Biol 13:25-33, 1995), and transgenic mice overexpressing the Thr164 or Ile164 receptors in the heart (Turki et al., Proc. Nat. Acad. Sci., USA 93:10483-10488, 1996). In addition, a synonymous polymorphism of C or A at nucleotide 523 in the coding sequence has been reported to be associated with altered responsiveness to salbutamol in Japanese families (Ohe, M. et al., Thorax 50:353-359, 1995).
In addition to the above polymorphisms in the coding block, several SNPs in the 5xe2x80x2 promoter region have recently been identified and are located at nucleotides xe2x88x921023 (A or G), xe2x88x92654 G or A), xe2x88x92468 (C or G), xe2x88x92367 (T or C), xe2x88x9247 (C or T) and xe2x88x9220 (T or C) (Scott, M. G. H. et al., Br J Pharmacol 126:841-844, 1999). Thus, eleven polymorphic sites have previously been identified in the region of the xcex22AR gene located between nucleotides 565 and 2110 of GenBank Accession No. M15169.1 (see FIG. 1 (SEQ ID NO:1)).
Messenger RNA transcripts of the xcex22AR gene have a 5xe2x80x2 leader region harboring a short open reading frame (ORF), termed the xcex22AR 5xe2x80x2-leader cistron (5xe2x80x2LC), that encodes a 19 amino acid peptide (Kobilka, B. K. et al., J. Biol. Chem.: 262:7321-7327, 1996). This xcex22AR upstream peptide (BUP) modulates translation of xcex22AR mRNA, and thereby regulates cellular expression of the receptor (Parola, A. L. et al., J Biol Chem 269:4497-4505, 1994). The polymorphic site located at xe2x88x9247, described above, is in this 5xe2x80x2LC and results in either Arg or Cys being encoded at the terminal amino acid position 19) of the BUP. It was recently reported that the Cys19 variant of the BUP is associated with greater xcex22AR expression than the Arg19 BUP variant (McGraw et al., J. Clin. Invest. 102:1927-1932, 1998).
Several groups have suggested associations between some of the above xcex22AR amino acid variants and increased susceptibility to various conditions, including: high blood pressure (Gly16 variant, Hoit et al., Am Heart J 139:537-542, 2000; Gratze et al., Hypertension 33:1425-1430, 1999 and Kotanko, P. et al., Hypertension 30:773-776, 1997; cf. Arg16 variant, Busjahn et al., Hypertension 35:555-560, 2000); atopy (Gly16 variant, Dewar et al., Clin. Exp. Allergy 28:442-448, 1998); nocturnal asthma (Gly16 variant, Turki et al., J. Clin. Invest. 95:1635-1641, 1995); response to treatment for obesity (Gly16 variant, Sakane et al., Lancet 353:1976, 1999); myasthenia gravis (Arg16 variant, Xu, B. -Y. et al., Clin. and Exp. Immunol. 119:156-160, 2000); childhood asthma (Gln27 variant, Dewar et al., J. Allergy Clin. Immun. 100:261-265, 1997); obesity (Glu27 variant, Large et al., J. Clin. Invest. 100:3005-3013, 1997); and mortality from congestive heart failure (Ile164 variant, Liggett et al., J. Clin. Invest. 102:1534-1539, 1998).
Several of the polymorphic sites (PS) in the xcex22AR gene have been reported to be in linkage disequilibrium with each other, including between the +46 and +79 PS (Martinez et al., J. Clin. Invest. 100:261-265, 1997; Dewar et al., supra), between the xe2x88x9247, +46 and +79 PS (McGraw et al., supra), between the xe2x88x9247, xe2x88x9220, +46, and +79 PS (Yamada et al., J. Clin. Endocrinol. Metab. 84:1754-1757, 1999), and between the +79 and +523 PS (Dewar et al., Clin. and Exp. Allergy 28:442-448, 1998). In addition, associations between various in vivo phenotypes and haplotypes for various combinations of these polymorphic sites have been suggested: obesity and a xe2x88x9247 C/xe2x88x9220 C haplotype (Yamada et al., supra), asthma severity and a +46G/+79G haplotype (which encodes the Gly16/Gln27 variant) (Weir et al., Am J. Resp. Crit Care Med. 158:787-791, 1998); bronchial hyperresponsiveness (BHR) and a +46 G/+79 G haplotype (D""amato et al., Am. J. Resp. Crit. Care Med. 158:1968-1973, 1998); hypertension and a xe2x88x9247 T/+46 A/+79 C haplotype, and the following expanded version thereof: xe2x88x921023 G/xe2x88x92654 A/xe2x88x9247 T/xe2x88x9220 C/+46 A/+79 C (Timmerman et al. Kidney Int. 53:1455-1460, 1998; WO 99/37761). An association between reduced xcex22AR promoter activity in vitro and a haplotype of xe2x88x92468 G/xe2x88x92367 C/xe2x88x9247 C/xe2x88x9220 C has also been reported (Scott et al., Br. J. Pharmacol. 126:841-844, 1999). However, no haplotypes covering more than six of the above 11 sites have been reported.
It has also been suggested that some of the xcex22AR gene polymorphisms discussed above may act as disease modifiers in asthma or may be the basis for the known interindividual variation in the bronchodilating response to xcex2-agonists (Liggett, S. B. xe2x80x9cThe genetics of xcex22-adrenergic receptor polymorphisms: relevance to receptor function and asthmatic phenotypes.xe2x80x9d in: Liggett, S. B. and Meyers, D. A., The Genetics of Asthma (1996) pp. 455-478). Indeed, two groups have reported that individuals homozygous or heterozygous for the Arg16 variant are more likely to respond to albuterol than individuals homozygous for the Gly16 variant. (Martinez, F. D. et al., J Clin Invest 100:3184-3188, 1997 and Lima, J. J., et al., Clin Pharmacol Ther 65:519-525, 1999). It has also been reported that asthmatic individuals who are homozygous for the Arg16 variant are more likely to exhibit decreased response to repeated use of albuterol (Drazen et al., WO 98/39477). Interestingly, another group reported bronchodilator desensitization in asthmatics homozygous for the Gly16 variant following continuous therapy with the xcex2-agonist formoterol (Tan et al., Lancet 350:995-999, 1997). Other studies failed to demonstrate any correlations between adverse drug response and regular treatment with xcex2-agonists (Hancox, R. J. et al., Eur Respir J 11:589-593, 1998; Lipworth, B. J. et al., Clinical Science 96:253-259, 1999). Moreover, none of the human physiologic studies assessed the relevance of haplotypes of multiple xcex22AR polymorphisms in both the promoter and coding regions for predicting the bronchodilator response to xcex2-agonists. Also, the phylogeny of these haplotypes and their distribution amongst different ethnic groups, which has particular relevance to pharmacogenetics, has not been explored.
Because of the potential for individual polymorphisms and haplotypes in the xcex22AR gene to affect susceptibility to a number of diseases, as well as affect response to xcex2-agonist therapy, it would be useful to determine whether additional polymorphisms exist in the xcex22AR gene, how such polymorphisms are combined in different copies of the gene to (haplotypes), and whether the frequencies of such polymorphisms and haplotypes vary among different ethnic groups. Such information would be useful for studying the biological function of xcex22AR as well as in identifying drugs targeting xcex22AR for the treatment of disorders related to its abnormal expression or function.
Accordingly, the inventors herein have discovered two novel polymorphic sites in the xcex22AR gene. These polymorphic sites (PS) correspond to the nucleotide positions 879 and 1182 in the promoter region of the xcex22AR gene (see FIG. 1) and are designated PS2 and PS5, respectively, to reflect their order in the xcex22AR gene relative to the other 11 polymorphic sites (Table 3). The polymorphisms at these sites are cytosine or adenine at PS2 and cytosine or thymine at PS5. It is believed that xcex22AR-encoding polynucleotides containing one or more of these novel polymorphic sites will be useful in studying the expression and biological function of xcex22AR, as well as in developing drugs targeting this receptor.
In addition, the inventors have determined the identity of the alternative nucleotides present at these sites, and at the previously identified 11 polymorphic sites described above (see Table 3), in a human reference population of apparently normal unrelated individuals representing four major population groups and in a cohort of asthma patients. The inventors herein have also identified how the polymorphisms at these 13 polymorphic sites in the xcex22AR gene (see FIG. 1) are combined into haplotypes in the reference and patient populations (see Tables 4 and 5) and discovered that certain pairs of these haplotypes, designated 2/2, 2/4, 2/6, 4/4 and 4/6 are predictive of bronchodilator response to albuterol. Asthmatic patients having xcex22 haplotype pairs 2/2 and 4/6 respond well to albuterol while patients with xcex22AR haplotype pairs 2/4 and 4/4 exhibit little to no response. xcex22AR haplotype pair 2/6 is associated with a moderate bronchodilator response. The inventors herein have also discovered that the presence of one of these medically significant haplotype pairs in an asthma patient may be predicted with high confidence by genotyping only three sites: PS3, PS9 and PS11.
Thus, in one embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence which is a polymorphic variant of a reference sequence for the xcex22AR gene or a fragment thereof. The reference sequence comprises SEQ ID NO:1 and the polymorphic variant comprises at least one polymorphism selected from the group consisting of adenine at PS2 and thymine at PS5. A particularly preferred polymorphic variant is a naturally-occurring isoform (also referred to herein as an xe2x80x9cisogenexe2x80x9d) of the xcex22AR gene. A xcex22AR isogene of the invention comprises guanine or adenine at PS1, cytosine or adenine at PS2, guanine or adenine at PS3, guanine or cytosine at PS4, cytosine or thymine at PS5, cytosine or thymine at PS6, cytosine or thymine at PS7, thymine or cytosine at PS8, adenine or guanine at PS9, cytosine or guanine at PS10, guanine or adenine at PS11, cytosine or thymine at PS12, and cytosine or adenine at PS13. The invention also provides a collection of xcex22AR isogenes, referred to herein as a xcex22AR genome anthology.
A xcex22AR isogene may be defined by the combination and order of these polymorphisms in the isogene, which is referred to herein as a xcex22AR haplotype. Thus, the invention also provides data on the number of different xcex22AR haplotypes found in the above four population groups. This haplotype data is useful in methods for deriving a xcex22AR haplotype from an individual""s genotype for the xcex22AR gene and for determining an association between a xcex22AR haplotype and a particular trait.
In another embodiment, the invention provides a recombinant expression vector comprising one of the polymorphic xcex22AR genomic variants operably linked to expression regulatory elements as well as a recombinant host cell transformed or transfected with the expression vector. The recombinant vector and host cell may be used to express xcex22AR for protein structure analysis and drug binding studies.
The invention also provides methods, compositions, and kits for haplotyping and/or genotyping the xcex22AR gene in an individual. In one embodiment, the genotyping method comprises isolating from the individual a nucleic acid mixture comprising the two copies of the xcex22AR gene present in the individual and determining the identity of the nucleotide pair at one or both of PS2 and PS5 in the two copies to assign a xcex22AR genotype to the individual. In another embodiment, a method for predicting an individuals haplotype pair comprises determining the individuals genotype at PS3, PS9 and PS11. The compositions contain oligonucleotide probes or primers designed to specifically hybridize to one or more target regions containing, or that are adjacent to, one or both of PS2 and PS5. Kits of the invention comprise a set of oligonucleotides for genotyping at least PS3, PS9 and PS11 and may also comprise additional oligonucleotides for genotyping one or more additional polymorphic sites selected from the group consisting of PS1, PS2, PS4, PS5, PS6, PS7, PS8, PS10, PS12 and PS13. The methods and compositions for genotyping or haplotyping PS2 and PS5 in the xcex22AR gene are useful for studying the effect of the alternative nucleotides at PS2 and PS5 in the etiology of various diseases and efficacy of drugs targeting the xcex22AR. Methods and kits for genotyping and haplotyping PS3, PS9 and PS11 are useful for predicting an asthmatic patient""s bronchodilating response to xcex2-agonists.
In yet another embodiment, the invention provides a method for identifying an association between a xcex22AR haplotype and a trait. In preferred embodiments, the trait is susceptibility to a disease, disease severity, the staging of a disease or response to a drug. Such methods have applicability in developing diagnostic tests and therapeutic treatments for one or more conditions selected from the group consisting of congestive heart failure, arrhythmia, ischemic heart disease, hypertension, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes, myasthenia gravis (MG) and premature labor. In other preferred embodiments, the drug is an agonist or antagonist of xcex22AR.
The present invention also provides genetically modified animals comprising one or more of the novel xcex22AR genomic polymorphic variants described herein and methods for producing such animals. Such animals are useful for studying expression of the xcex22AR isogenes in vivo, for in vivo screening and testing of drugs targeted against xcex22AR protein, and for testing the efficacy of therapeutic agents and compounds targeting the xcex22AR in a biological system.
The present invention also provides a computer system for storing and displaying polymorphism data determined for the xcex22AR gene. The computer system comprises a computer processing unit; a display; and a database containing the polymorphism data. The polymorphism data includes the polymorphisms, the genotypes and the haplotypes identified for the xcex22AR gene in one or both of the reference population and the patient population. In a preferred embodiment, the computer system is capable of producing a display showing xcex22AR haplotypes organized according to their evolutionary relationships.
Another aspect of the invention is based on the discovery of novel information relating to linkage disequilibrium in Caucasians between PS1, PS3, PS4 and PS6 and the xcex22AR polymorphic sites previously reported to be associated with various medical conditions, i.e., PS9 and PS10. Thus, the present invention also provides a method for predicting a Caucasian individual""s genetic predisposition to any disease or condition known to be associated with one of the alternative alleles at PS9 or PS10 in the xcex22AR gene. The method comprises determining a first genotype for a first polymorphic site in the individual""s xcex22AR gene, wherein the first polymorphic site is selected from the group consisting of PS1, PS3, PS4 and PS6, and using the first genotype to predict a second genotype for a second polymorphic site in the individual""s xcex22AR gene, wherein if the first polymorphic site is PS1, PS4 or PS6, then the second polymorphic site is PS10, and if the first polymorphic site is PS3, then the second polymorphic site is PS9. In a preferred embodiment, the disease or condition is selected from the group consisting of atopy, nocturnal asthma, childhood asthma, hypertension, obesity, response to treatment for obesity and MG.
The present invention further provides methods for predicting an asthma patient""s response to xcex2-agonist therapy. In one embodiment, the method comprises determining the genotype for the patient at PS3, PS9 and PS11, wherein the patient is likely to exhibit a good response to a standard dose of the xcex2-agonist if the patient is homozygous for guanine at each of PS3, PS9 and PS11 or if the patient is heterozygous A/G at each of PS3, PS9 and PS11. If the patient is homozygous A/A/G at PS3, PS9 and PS11, respectively, then the patient is likely to not respond to standard dosages of the xcex2-agonist. In a preferred embodiment, the xcex2-agonist is albuterol. Thus, knowledge of a patient""s xcex22AR genotype for PS3, PS9 and PS11 provides a physician with information useful for making determinations as to which drug to administer and dosages of the drug.